Schnurri-3 inhibition rescues skeletal fragility and vascular skeletal stem cell niche pathology in a mouse model of osteogenesis imperfecta

Osteogenesis imperfecta (OI) is a disorder of low bone mass and increased fracture risk due to a range of genetic variants that prominently include mutations in genes encoding type collagen. While it is well known that OI reflects defects in the activity of bone-forming osteoblasts, it is currently unclear whether OI also reflects defects in the many other cell types comprising bone, including defects in skeletal vascular endothelium or the skeletal stem cell populations that give rise to osteoblasts and whether correcting these broader defects could have therapeutic utility. Here, we find that numbers of skeletal stem cells (SSCs) and skeletal arterial endothelial cells (AECs) are augmented in Col1a2oim/oim mice, a well-studied animal model of moderate to severe OI, suggesting that disruption of a vascular SSC niche is a feature of OI pathogenesis. Moreover, crossing Col1a2oim/oim mice to mice lacking a negative regulator of skeletal angiogenesis and bone formation, Schnurri 3 (SHN3), not only corrected the SSC and AEC phenotypes but moreover robustly corrected the bone mass and spontaneous fracture phenotypes. As this finding suggested a strong therapeutic utility of SHN3 inhibition for the treatment of OI, a bone-targeting AAV was used to mediate Shn3 knockdown, rescuing the Col1a2oim/oim phenotype and providing therapeutic proof-of-concept for targeting SHN3 for the treatment of OI. Overall, this work both provides proof-of-concept for inhibition of the SHN3 pathway and more broadly addressing defects in the stem/osteoprogentior niche as is a strategy to treat OI.


Introduction
Osteogenesis imperfecta (OI) is a disorder with heterogeneous genetic causes that prominently include mutations in the type I collagen genes, COL1A1 and COL1A2. The hallmark of OI is low bone mass and skeletal fragility, resulting in susceptibility to fracture 1,2 . Currently, treatments including anti-resorptive drugs bisphosphonates (e.g. alendronate and zoledronate) are used for severe OI, though their e cacy in reducing fracture rates remains under investigation [3][4][5][6][7][8][9][10][11] . Bone anabolic agents, including are also under investigation for their potential bene t to OI patients in both preclinical and clinical trials 5,6 . Experimental therapies inhibiting TGF signaling are also under study for treatment of OI 12 . Despite these advances and ongoing investigation there is still not a standard of care that has been clearly established to reduce fracture rates in OI and therefore a remaining unmet need for OI treatments. Whereas most therapeutic efforts in OI directly target well-established directly regulators of osteoblast or osteoclast activity, we hypothesized that skeletal microenvironmental dysregulation may also be a core feature of OI pathogenesis in addition to osteoblast intrinsic defects. Thus, targeting pathways that can both rescue these microenvironmental defects and cell intrinsic defects in osteoblast activity may provide a new and effective strategy for OI treatment.
Along these lines, it is increasingly appreciated that ancillary tissue types such as vascular endothelium present in bone actively contribute to osteogenesis 13,14 . Though it is known that deposition of collagen type I is critical for coupling between angiogenesis and osteogenesis during skeletal development, the degree to which the vascular microenvironment of bone is dysregulated in OI is unclear 15,16 . It is also unknown how OI disrupts the early stem and progenitor cellular compartment in bone, especially a recently identi ed population of cells displaying formal evidence of stemness, SSCs that are de ned through multicolor ow cytometry as lineage-Thy1-6C3-alpha-v integrin + CD200 + CD105-cells 17,18 .
In considering alternative approaches to augment bone formation and correct any potential vascular microenvironmental defects in OI, Schnurri-3 (SHN3, HIVEP3), a critical negative regulator of bone formation in both mice and humans offers an attractive novel approach 19,20 . Mice lacking SHN3 display an osteosclerotic phenotype with profoundly augmented osteoblast activity leading to near absolute protection from age-related bone loss. In addition to the ability of SHN3 de ciency to directly drive the intrinsic bone formation activity of osteoblasts, SHN3 de ciency also acts in osteoblasts to control the skeletal vascular microenvironment by regulating production of a recently described osteoblast derived angiogenic factor, SLIT3 14,21 . Consistent with this, SHN3 de cient mice (Shn3 -/-) mice display both enhanced bone formation and increased amounts of skeletal vascular endothelium, and both of these phenotypes are SLIT3 dependent 14 . Thus, the SHN3/SLIT3 signaling axis in osteoblasts offers a potential method to not only increase bone formation but also to address microenvironmental disruption occurring in skeletal disorders.
Here we sought to determine whether skeletal microenvironmental dysfunction occurs in OI and how this may impact early SSC populations, using a widely studied OI mouse model, Col1a2 oim/oim mice (OIM mice) displaying spontaneous fractures 22 . We further nd evidence that correcting these defects in the skeletal vascular microenvironment can ameliorate the OI phenotype, as SHN3-de ciency can rescue the Col1a2 oim/oim model, correcting both the dysregulation in the SSC vascular niche and skeletal fragility in the Col1a2 oim/oim model.

Results
Altered skeletal vascular composition in Col1a2 oim/oim mice Though deposition of collagen type I has been suggested to mediate coupling between angiogenesis and osteogenesis during bone formation, the degree to which disruption of this osteo-angio microenvironment contributes to OI and the speci c forms of vascular endothelium that are impacted remain unclear 14,23 .
To address this, we selected Col1a2 oim/oim mice as a model for human OI. As anticipated, Col1a2 oim/oim mice showed a severe osteopenic phenotype and spontaneous bone fractures relative to littermate controls in 4 weeks old (Fig. 1A-B). To avoid potential confounding due to the presence of fracture healing responses, we selected adolescent Col1a2 oim/oim mice without spontaneous fractures for further examination.
Of note, a series of recent studies revealed that skeletal endothelial cells can be further divided into two separate subpopulations, arterial endothelial cells (AECs) and sinusoidal endothelial cells (SECs) and each of which play a distinct role in supporting osteogenesis. We then established a new multi-color ow cytometry using the combination of podoplanin (PDPN) and Sca-1 to distinguish AECs from SECs in the marrow cavity (Fig. 1C). Based on this, the population of AECs but not SECs was signi cantly elevated in bones of Col1a2 oim/oim mice (Fig. 1D). Immuno uorescence analysis con rmed that the abundance of arterial vessels was indeed increased substantially in Col1a2 oim/oim mice (Fig. 1E). Of note, this effect was speci c for AEC, as other forms of skeletal endothelium, including CD31 hi endomucin hi (EMCN hi ) endothelial cells 23 , where not altered ( Fig. S1A-B). Thus, expansion of AEC is a feature of OI models.
Elevated abundance of skeletal stem cells in Col1a2 oim/oim mice Skeletal stem cells (SSCs) serve as the ultimate source of all bone forming osteoblasts, therefore perturbations in the SSC compartment are likely central to many skeletal disorders 24,25 . Considering that AECs regulate hematopoietic stem cell (HSC) proliferation 26 , we then considered whether the AEC expansion seen in OI may similarly translate to SSC alterations. To evaluate this, we utilized an established multi-color ow cytometry panel identifying SSCs 18 to analyze skeletal stem/progenitor cells populations in long bones of Col1a2 oim/oim mice ( Fig. 2A). Interestingly, the abundance of immunophenotypic SSCs was elevated in Col1a2 oim/oim mice, yet the amounts of pre-bone cartilage skeletal progenitors (pre-BCSPs) and bone cartilage skeletal progenitors (BCSPs), SSC-derived non-stem progenitors 17 , was unchanged (Fig. 2B). This, alteration in the ratio of different stages of SSC maturation indicates that OI impacts the stem cell differentiation hierarchy. Consistent with this, the number of CD200 positive cells was obviously increased in the primary spongiosum near the growth plate in Col1a2 oim/oim mice, a region characterized as housing more SSCs and more artery/arterioles 16,17,27,28 (Fig. 2C). Moreover, delayed osteogenesis attributed to impaired mineralization was observed in Col1a2 oim/oim mice evidenced by the whole-mount skeletal staining in neonatal mice (Fig. 2D), with the early emergence of this phenotype being consistent with alterations in the early SSC compartment. Given increase in both SSCs and AECs seen in Col1a2 oim/oim mice, disruptions in an angiogenic SSC niche are likely contributors to the overall OI skeletal phenotype.  Fig. 2A-B). Moreover, expression of genes related to stemness and early osteogenesis and endochondral ossi cation were also decreased in SSCs derived from Col1a2 oim/oim mice (Fig. 3E). To determine if these transcriptional alterations in SSCs translate into cell-intrinsic functional differences, we sorted and transplanted the equal amounts of SSCs into kidney capsule for an organoid bone formation assay. µCT analysis showed that Col1a2 oim/oim SSCs display functional defects in mineralization (Fig. 3F). Taken together, these ndings implicate a broader set of cellular pathology, including defects in SSCs and AECs, beyond functional defects in mature osteoblasts in OI.
Deletion of SHN3 improves bone properties in Col1a2 oim/oim mice There remains a substantial unmet clinical need for OI treatments that reduce fracture risk in OI 11,29 . We have previously reported that SHN3 acts as a cell intrinsic negative regulator of both osteoblast bone formation activity and the ability of osteoblasts to promote an osteoanabolic vascular microenvironment in bone 14 . To evaluate both whether SHN3 inhibition is a potential therapeutic approach to treat OI and also whether SHN3-mediated regulation of the skeletal vascular microenvironment is relevant to OI phenotypes, we intercrossed Shn3 -/mice with Col1a2 oim/oim mice 14 . Ablation of SHN3 19,30 provided a complete or near complete reversal of the low bone mass observed in both trabecular and cortical bone compartments in Col1a2 oim/oim mice (Fig. 4A, B). Histomorphometric analysis revealed that SHN3de ciency was su cient to reverse the osteopenic phenotype and attenuated osteoblast numbers in Col1a2 oim/oim mice (Fig. 4C, D). Likewise, the decrease in bone formation rate (Fig. 4E) observed in Col1a2 oim/oim mice was normalized through additional deletion of Shn3 (Fig. 4F, G). Thus, deletion of Shn3 is capable to block OI-induced bone loss by normalizing bone remodeling.
Ablation of Shn3 prevents spontaneous fractures in Col1a2 oim/oim mice While it is encouraging that SHN3-de cient Col1a2 oim/oim mice display a restoration of bone formation parameters, the most clinically relevant endpoint is the prevention of fractures, the major source of morbidity in clinical OI. Indeed, Col1a2 oim/oim mice displayed spontaneous fractures starting at 3 weeks of age that increased in frequency until an average of 3 fractures per mouse could been seen at 8 weeks of age ( Fig. 5A-C). Strikingly, we found SHN3 de ciency is able to completely prevent the spontaneous bone fractures in Col1a2 oim/oim mice as no fractures occurred in Shn3 −/− Col1a2 oim/oim mice. This protection from fracture in Shn3 −/− Col1a2 oim/oim mice also correlated with rescue of the running seen in Col1a2 oim/oim mice, perhaps re ecting the effect of fracture-associated stress on skeletal growth. Thus, SHN3 de ciency not only normalizes bone formation in Col1a2 oim/oim mice but completely prevents the signature spontaneous fractures occurring in this OI model. SHN3 de ciency corrects the vascular and SSC compositional changes in Col1a2 oim/oim mice We next evaluated whether the ability of SHN3-de ciency to rescue the spontaneous fractures in Col1a2 oim/oim mice re ected a correction of the skeletal microenvironment. To this end, we analyzed the cellular composition of the vascular endothelium and SSC compartments in Shn3 -/-Col1a2 oim/oim mice using a previously reported multi-color ow cytometry panel 17 . Strikingly, Shn3 de ciency rescued the skeletal vascular pathology in Col1a2 oim/oim mice. Shn3-depletion attenuated the AEC expansion seen in Col1a2 oim/oim mice while not impacting the amount of SECs (Fig. 6A-B). Interestingly, SHN3 de ciency alone did not notably alter the amount of SSCs or their production of downstream cell types. Despite this, Shn3-de ciency reversed the SSC expansion seen in Col1a2 oim/oim mice, limiting the hyper proliferative phenotype seen in the SSC-enriched primary spongiosum region in Col1a2 oim/oim mice adjacent to the growth plate (Fig. 6C-E). These results implied that the expansion of SSCs is closely linked to the AECs expansion seen in Col1a2 oim/oim mice, in line with emerging evidence that endothelial cells play a critical role in the SSC niche 31 . Thus, deletion of SHN3 corrected the cellular alterations in AECs and SSCs seen in Col1a2 oim/oim mice, correcting the cellular perturbations in seen with this OI model (Fig. 6A, C). This nding also links correction of these cellular pathologies with overall rescue of the OI phenotype.
Shn3 -silencing is a candidate therapeutic approach for OI AAV-based gene therapy is emerging as an attractive modality for the treatment of skeletal disorders due to its ability to potentially mediate long-lasting effects after a single treatment and the ability to address therapeutic targets that are challenging for traditional small molecule or biologic therapies, such as Shn3 [32][33][34] . Previous studies utilized AAV serotype-9 to deliver a Shn3-silencing construct that mediated a robust reduction in Shn3 expression in osteoblastic lineage cells and accordingly augmented bone mass under both baseline physiologic conditions and in a mouse model of post-menopausal osteoporosis. 35 To investigate whether a gene therapy approach to inhibit SHN3 expression is capable of treating OI, we constructed replication-defective recombinant AAV serotype 9 (rAAV9) harboring enhanced green uorescent protein (EGFP)-expressing plasmids that additionally bear either a Shn3 targeting arti cial microRNA (rAAV9-amiR-Shn3) or a miRNA-control (rAAV9-amiR-Ctrl) as previously described 35 . This vector displayed robust tropism of for osteoblasts and favorable relative speci city of payload delivery after intraarticular injection in prior studies 35 . We administered rAAV9-amiR-Shn3 or rAAV9-amiR-Ctrl to Col1a2 oim/oim mice via intraarticular injection at 4 weeks of age into contralateral limbs of the same mouse and evaluated their skeletal phenotype at 12 weeks of age (Fig. 7A). Through IVIS optical imaging, expression of eGFP was predominantly localized to the hindlimb of rAAV9-injected mice, with both the femur and tibia displaying high intensity eGFP expression (Fig. 7B-C). Fluorescence microscopy con rmed that bone-lining osteoblasts were effectively transduced by the rAAV9 vector (Fig. 7D). Furthermore, trabecular bone volume was signi cantly higher in rAAV9-amiR-Shn3 administered limbs compared to contralateral rAAV9-amiR-Ctrl limbs and cortical bone is also signi cantly thickened, which reduces the probability of fractures (Fig. 7E-F). This provides proof-of-concept that postnatal therapeutic targeting of Shn3 is able to reverse the osteopenia seen in the Col1a2 oim/oim mice and provides speci c demonstration of an AAV-based gene therapy approach to Shn3 targeting.

Discussion
Currently, there remains a substantial unmet clinical need for methods to treat OI, as it whether antiresorptive drugs such as bisphosphonates or other repurposed osteoporosis therapies will correct the increased fracture rate that is the signature clinical issue of OI remains an area of active investigation. In part this unmet need is a call for both an improved mechanistic understanding of OI and for investigation into innovative means to correct or compensate for the cellular, architectural and bone materials properties de cits driving skeletal fragility in OI. The functional and physical coupling between osteogenesis and angiogenesis is increasingly emerging as a critical point of dysfunction in skeletal disorders and also a promising but largely unexplored therapeutic opportunity. Previously, we found that SHN3 acts in osteoblasts to regulate production of SLIT3, which in turn acts as a skeletal speci c angiogenic factor 14 . Through this angiogenic activity, SLIT3 in turn creates a skeletal vascular microenvironment that enables anabolic bone formation. We here nd a linkage between AEC abundance and SSC expansion in OI that in turn suggests a broader functional linkage between AECs and SSCs, perhaps with AECs serving as part of the SSC niche. This ts with an emerging picture that speci c skeletal progenitor populations may be speci cally localized to perivascular or peri-arteriolar regions, and provides some of the rst direct evidence that vascular modulation produces corresponding changes in SSCs as functional evidence for a perivascular SSC niche 31,38,39 . This also raises the question of whether SLIT3 is a speci c regulator of SSC abundance through a selective ability to modulate AECs as opposed to SECs, suggesting a model whereby the composition of skeletal vascular endothelium can be tuned by a series of subset vessel-type speci c angiogenic factors, possibly including PDGF-bb, VEGF isoforms or others, that in turn govern the composition of the pool of early skeletal progenitors 40 . In this manner, a vascular targeted osteoanabolic agent may be synergistic with a traditional osteoblast targeted osteoanabolic through activity to "prime" the osteoanabolic effect both by preparing the pool of early vasculature associated stem and progenitor cells and also by creating a microenvironment that favors bone formation. Thus, the SHN3-targeting gene therapy approach taken here is also anticipated to be complimentary to either established osteoanabolic or antiresorptive therapeutics or to emerging therapeutic approaches that speci cally target the molecular pathogenesis of OI, such as anti TGF-β antibodies 5,12 .

Recent advances in single cell transcriptome analysis and the FACS-based de nition of skeletal cell types
Whether achieved via a gene therapy-based approach as tested here or via other methods, inhibition of SHN3 is an attractive approach for treating OI. Prior mechanistic studies have shown that, in addition to the above ability of SHN3 to regulate the bone vascular microenvironment through regulation of SLIT3 secretion, SHN3 mediates a cell intrinsic effect in osteoblasts to suppress bone formation. This occurs in part via the ability of SHN3 to suppress ERK-mediated phosphorylation of selected substrates 19,30 . While SHN3 is broadly expressed, the phenotypes associated with SHN3 de ciency appear to be limited to the skeleton, which is promising for the possibility that SHN3-targeted therapies will display a favorable effect to toxicity pro le.
In summary, this project has identi ed new cellular features of OI in the SSC and vascular compartments of bone and identi ed preclinical evidence supporting a new therapeutic approach centering on inhibition of the SHN3 pathway with an AAV delivered payload. We anticipate that this will not only motivate further development of AAV and SHN3-based gene therapeutic approaches, but moreover provide evidence for marked disruption in the vascular and SSC compartments as a feature of OI that could be central to disease pathogenesis.

Animals
Col1a2 oim/oim mice were obtained from the Jaxson Laboratory (B6C3Fe a/a-Col1a2 oim /J, Stock No: 001815, Bar Harbor, ME, USA); Shn3 −/− mice were described in our previous studies 14,19 . Dual heterozygous Shn3 +/− Col1a2 oim/+ mice were used for breeding to generate Shn3 +/+ Col1a2 +/+ mice, Shn3 +/+ Col1a2 oim/oim mice, Shn3 −/− Col1a2 +/+ mice and Shn3 −/− Col1a2 oim/oim mice. All mice were housed up to four per cage under a 12-hour light-dark cycle with chow ad libitum in the Laboratory Animal Center at the Xiamen University. All mouse experiments were handled according to the protocols approved by the Institutional Animal Care and Use Committee of Xiamen University Laboratory Animal Center.

Radiography and micro-CT analysis
Whole-body radiographs of experimental mice were captured by a Faxitron X-ray system. We de ned fractures in the humerus, forearms, femurs and tibias by bone deformity and callus formation. We performed µCT scanning using a µCT 35 system (Scanco Medical, Sweden) at the Weill Cornell-Citigroup Biomedical Imaging Core according to the parameters in our previous study 21 . The analysis was conducted by a technician blinded to the genotypes of the mice under analysis.

Histology and dynamic histomorphometry
We injected the experimental and control mice intraperitoneally with a dose of 20mg/kg calcein on days 1 and 5 before sacri ce for measurement of bone formation rate. Resin embedding and sectioning without decalci cation was performed for von Kossa staining, toluidine blue staining and TRAP staining as described in our previous study 41 . We then use the Osteomeasure System (OsteoMetrics, Atlanta, USA) for histomorphometric analysis as previously described 13 .
Flow cytometry analysis and cell sorting

Kidney capsule transplantation model
Kidney capsule transplantation models were performed as previously described 17 rAAV9-mediated silencing of Shn3, intra-articular injection Arti cial miRNA-containing plasmids targeting murine Shn3 were generous gifts from Dr. Guangping Gao. Replication-de cient recombinant AAV (rAAV) vector design and production were performed as previously described 35 . In brief, engineered amiR cassettes targeting Shn3 (amiR-Shn3) or control (amiR-ctrl) were constructed within vector plasmids between CB promoter and the reporter gene Egfp which enable visual tracking of transduced cells, or tissues. Plasmids containing amiR cassettes, AAV2/9 and helper plasmids were mixed and transfected into HEK293 cells using PEI-MAX 40000 (Polysciences, 24765-1) to generate rAAV for experimental use. These rAAV batches were then collected and puri ed following a traditional CsCl sedimentation protocol. The concentration of rAAV9-amiR-Shn3 and rAAV9-amiR-ctrl was then tittered as previously described (Yang YS, Nat Commun, 2020).
For local delivery of rAAV, intra-articular injections were performed when male-Col1a2 oim/oim mice or controls reached 1 month of age. To avoid confounding due to active fracture repair, Col1a2 oim/oim mice display radiographic evidence of fracture were excluded from the experimental cohort. After anesthesia and surgical site preparation, a 1-mm anterolateral skin incision was made above knee articular capsule.
A total volume of 5uL containing 1 × 10 12 GC rAAV9-amiR-shn3 or rAAV9-amiR-ctrl were injected into the articular capsule of contralateral hindlimb sti e/knee joints on the same host to allow for paired analysis of local effects in the same host. The needle was retained in the injection site for 2-5 minutes to avoid leakage. Incisions were then sutured and analgesic administration was performed as described previously 14 . 8 weeks after injection, individuals were subjected to in vivo IVIS optical imaging and lower limbs were dissected for downstream analysis.

Statistical analysis
All statistical analysis was performed using GraphPad Prism (v6.0a; GraphPad, La Jolla, CA, USA). A twotailed Student's t test was used to determine signi cance for comparison of only two groups. One-way ANOVA with Tukey's post-hoc tests were used to determine signi cance for comparisons between multiple groups. A P value < 0.05 indicated statistical signi cance. Error bars are presented as mean ± s.e.m.     SHN3-silencing is a candidate therapeutic approach for OI (A) Schematic diagram of strategy for rAAV9-amiR-ctrl or amiR-shn3 injected into knee joints in 4 weeks old Col1a2 oim/oim , rAAV9-amiR-ctrl in the left hindlimbs and amiR-shn3 in the right hindlimbs, sacri ced in 12 weeks old.